The Salmonella genomic island 1 is specifically mobilized in trans by the IncA/C multidrug resistance plasmid family

Background

The Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55.

Methodology/Principal Findings

Here, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency <10⁻⁹). In our collection, ESBL gene-carrying plasmids were mainly from the IncHI2 and I1 groups and thus were unable to mobilize SGI1. However, the horizontal transfer of SGI1 was shown to be specifically mediated by conjugative helper plasmids of the broad-host-range IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10⁻³ to 10⁻⁶ transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase bla _CMY-2 gene were shown to mobilize in trans SGI1.

Conclusions/Significance

The ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic resistance genomic island SGI1 and its close derivatives.     

Stability, entrapment and variant formation of Salmonella genomic island 1

Background

The Salmonella genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S. Typhimurium.

Methodology/Principal Findings

Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in Salmonella populations of 17 Salmonella enterica serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the E. coli chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.

Conclusions/Significance

Despite that excision of SGI1 from the chromosome was proven in SGI1⁺ Salmonella populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.     

IncC helper dependent plasmid-like replication of Salmonella Genomic Island 1

The Salmonella genomic island 1 (SGI1) and its variants are mobilized by IncA and IncC conjugative plasmids. SGI1-family elements and their helper plasmids are effective transporters of multidrug resistance determinants. SGI1 exploits the transfer apparatus of the helper plasmid and hijacks its activator complex, AcaCD, to trigger the expression of several SGI1 genes. In this way, SGI1 times its excision from the chromosome to the helper entry and expresses mating pore components that enhance SGI1 transfer. The SGI1-encoded T4SS components and the FlhDC-family activator proved to be interchangeable with their IncC-encoded homologs, indicating multiple interactions between SGI1 and its helpers. As a new aspect of this crosstalk, we report here the helper-induced replication of SGI1, which requires both activators, AcaCD and FlhDC_SGI1, and significantly increases the stability of SGI1 when coexists with the helper plasmid. We have identified the oriV_SGI1 and shown that S004-repA operon encodes for a translationally coupled leader protein and an IncN2/N3-related RepA that are expressed under the control of the AcaCD-responsive promoter P_S004. This replicon transiently maintains SGI1 as a 4–8-copy plasmid, not only stabilizing the island but also contributing to the fast displacement of the helper plasmid.     

Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids

Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3’ end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5’ end of dusA and the 3’ end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.     

Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

Summary The genome segment carrying the activities int and xis, responsible for integration and excision of phage 16-3, have been identified and cloned. Mutants were isolated, permitting the investigation of int, xis and att sites (attP, attR, attB) in trans arrangements. The efficiency and role of int- and xis-promoted reactions and of homologous recombination in the formation of lysogenic cells are established. The possible use of the cloned int-attP chromosomal segment in the manipulation of Rhizobium meliloti is discussed.     

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.Antimicrobial compounds have been used extensively in agriculture since the 1960s not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in fish, poultry, and other livestock (42). The risk of transferring antimicrobial drug resistance to nonresistant bacteria and the propagation of multidrug-resistant (MDR) bacteria from agricultural to clinical and/or community-associated settings are being debated by research, regulatory, and health authorities (27, 28). In this context, the recent discovery of a group of self-transferable IncA/C antimicrobial resistance plasmids, which are widely distributed among agricultural nontyphoidal Salmonella enterica isolates from the United States (24, 45) has caused considerable concern in the public health community. Similar IncA/C plasmids were identified in an MDR isolate from Madagascar of Yersinia pestis, the causative agent of the plague (16), and MDR strains of Vibrio cholerae O139 from China (34), as well as in MDR isolates of the fish pathogen Photobacterium damselae subsp. piscicida from the United States and Japan (21). While the IncA/C group of MDR plasmids seems to be efficient in collecting antimicrobial resistance traits and mobilizing them across geographical and taxonomical borders, little is known about the evolutionary origin of these plasmids or the genetic basis for their spread.The IncA/C reference plasmid, pRA1, was isolated in 1971 from the fish pathogen Aeromonas liquefaciens, later renamed Aeromonas hydrophila, as a transferable antimicrobial resistance plasmid conferring resistance to sulfonamides and tetracyclines (2). The repA gene of pRA1, located at the origin of replication and responsible for encoding the replication initiation protein A, has been sequenced (25) and is used for PCR-based replicon typing of IncA/C plasmids (7). repA genes from all sequenced IncA/C plasmids to date share at least 98% nucleotide sequence identity.To better understand the evolutionary origin of IncA/C plasmids, pRA1 was isolated, sequenced, and compared to all IncA/C plasmid sequences currently available. In addition to pRA1, a pRA1-derived cryptic IncA/C plasmid, designated pRAx, was also sequenced and included in the analysis. pRAx was isolated from Escherichia coli D7-3, a strain that was obtained through the conjugative transfer of pRA1 from A. hydrophila in 1980 (30). While the laboratory history of the pRAx-carrying strain E. coli D7-3 since the conjugative plasmid acquisition is unknown, pRAx was included in this study as it tested positive for the repA reference gene from pRA1 (100% nucleotide sequence identity) but negative for 11 out of 12 additional IncA/C marker genes that were shown to be part of a conserved plasmid backbone shared by recently isolated IncA/C plasmids (45).     

Initiation of synthesis of the structural proteins of Semliki Forest virus.

Insertion of phage λ DNA into the normal attachment site of the DNA of the host Escherichia coli has been studied by ultracentrifugation analysis of the conversion of covalent circles of F′450 (F′gal att^λ bio) to F′450(λ) circles. We have found that integration proceeds at the normal rate if, in addition to the int gene product and a proper combination of phage and bacterial attachment sites, a large pool of λ DNA and some activity of the excision gene xis are present. In addition, turnoff of both phage DNA synthesis and xis gene activity are required.     

Control of Staphylococcus aureus pathogenicity island excision

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15–17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small‐headed phage‐like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI‐encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI‐coded Xis is absolutely required for recombination between the SaPI att_L and att_R sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.     

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration,and Acquisition of the Plasmid Results in a Variable Cost of Fitness

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.     

Comparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coli

Increasing reports of multidrug resistance conferred by conjugative plasmids of Enterobacteriaceae necessitate a better understanding of their evolution. One such group is the narrow-host-range IncI1 plasmid type, known for their ability to carry genes encoding resistance to extended-spectrum beta lactamases. The focus of this study was to perform comparative sequencing of IncI1 plasmids from porcine enterotoxigenic Escherichia coli (ETEC), isolated irrespective of antimicrobial susceptibility phenotype. Five IncI1 plasmids of porcine ETEC origin and one IncI1 plasmid from a Salmonella enterica serovar Kentucky isolate from a healthy broiler chicken were sequenced and compared to existing IncI1 plasmid sequences in an effort to better understand the overall genetic composition of the IncI1 plasmid lineages. Overall, the sequenced porcine ETEC IncI1 plasmids were divergent from other sequenced IncI1 plasmids based upon multiple means of inferred phylogeny. High occurrences of IncI1 and IncA/C plasmid-associated genes and the bla_TEM and bla_CMY-2 beta lactamase genes were observed among porcine ETEC. However, the presence of bla_TEM and bla_CMY-2 did not strongly correlate with IncI1 plasmid possession, suggesting that these plasmids in porcine ETEC are not primarily associated with the carriage of such resistance genes. Overall, this work suggests a conservation of the IncI1 plasmid backbone among sequenced plasmids with a single locus for the acquisition of accessory genes, such as those associated with antimicrobial resistance. Furthermore, the high occurrence of IncI1 and IncA/C plasmids among clinical E. coli from commercial swine facilities is indicative of extensive horizontal gene transfer among porcine ETEC.     

Chromosomal transfer promoted by the promiscuous plasmid RP4.

We have studied the properties of the recombinant plasmid RP4λatt. This plasmid possesses the EcoRI-generated fragment of phage λ containing the genes att-int-xis (srIλ2–3) inserted into the single EcoRI site of the promiscuous plasmid RP4. The insertion of this λ fragment has no detectable effect on normal plasmid functions. However, it confers the ability to promote low-frequency polarized chromosomal transfer by int-promoted integration into the host λ attachment site attλ. We have succeeded in isolating an Hfr derivative which has the plasmid stably integrated at attλ. The Hfr derivative is unusual in having both an integrated and an autonomous RP4λatt stably coexisting in the same cell.     

pIMP-PH114 Carrying bla IMP-4 in a Klebsiella pneumoniae Strain is Closely Related to Other Multidrug-Resistant IncA/C2 Plasmids

The IncA/C plasmids are broad host-range vehicles which have been associated with wide dissemination of CMY-2 among Enterobacteriaceae of human and animal origins. Acquired metallo-β-lactamases (MBLs) such as the IMP-type enzymes are increasingly reported in multidrug-resistant Gram-negative bacteria worldwide, particularly in Enterobacteriaceae. We described the complete sequence of the first IMP-4-encoding IncA/C₂ plasmid, pIMP-PH114 (151,885 bp), from a sequence type 1 Klebsiella pneumoniae strain that was recovered from a patient who was hospitalized in the Philippines. pIMP-PH114 consists of a backbone from the IncA/C₂ plasmids, with the insertion of a novel Tn21-like class 1 integron composite structure (containing the cassette array bla _IMP-4-qacG-aacA4-catB3, followed by a class C β-lactamase bla _DHA-1 and the mercury resistance operon, merRTPCADE) and a sul2-floR encoding region. Phylogenetic analysis of the IncA/C repA sequences showed that pIMP-PH114 formed a subgroup with other IncA/C plasmids involved in the international spread of CMY-2, TEM-24 and NDM-1. Identical bla _IMP-4 arrays have been described among different Enterobacteriaceae and Acinetobacter spp. in China, Singapore and Australia but the genetic context is different. The broad host range of IncA/C plasmids may have facilitated dissemination of the bla _IMP-4 arrays among different diverse groups of bacteria.     

Antimicrobial Resistance,Class 1 Integrons,and Genomic Island 1 in Salmonella Isolates from Vietnam

Background

The objective was to investigate the phenotypic and genotypic resistance and the horizontal transfer of resistance determinants from Salmonella isolates from humans and animals in Vietnam.

Methodology/Principal Findings

The susceptibility of 297 epidemiologically unrelated non-typhoid Salmonella isolates was investigated by disk diffusion assay. The isolates were screened for the presence of class 1 integrons and Salmonella genomic island 1 by PCR. The potential for the transfer of resistance determinants was investigated by conjugation experiments. Resistance to gentamicin, kanamycin, chloramphenicol, streptomycin, trimethoprim, ampicillin, nalidixic acid, sulphonamides, and tetracycline was found in 13 to 50% of the isolates. Nine distinct integron types were detected in 28% of the isolates belonging to 11 Salmonella serovars including S. Tallahassee. Gene cassettes identified were aadA1, aadA2, aadA5, bla _PSE-1, bla _OXA-30, dfrA1, dfrA12, dfrA17, and sat, as well as open reading frames with unknown functions. Most integrons were located on conjugative plasmids, which can transfer their antimicrobial resistance determinants to Escherichia coli or Salmonella Enteritidis, or with Salmonella Genomic Island 1 or its variants. The resistance gene cluster in serovar Emek identified by PCR mapping and nucleotide sequencing contained SGI1-J3 which is integrated in SGI1 at another position than the majority of SGI1. This is the second report on the insertion of SGI1 at this position. High-level resistance to fluoroquinolones was found in 3 multiresistant S. Typhimurium isolates and was associated with mutations in the gyrA gene leading to the amino acid changes Ser83Phe and Asp87Asn.

Conclusions

Resistance was common among Vietnamese Salmonella isolates from different sources. Legislation to enforce a more prudent use of antibiotics in both human and veterinary medicine should be implemented by the authorities in Vietnam.     

Physical study of prophage excision and curing of lambda prophage from lysogenic Escherichia coli

A sex factor, F′450(λ), which can be isolated as a covalent circle of DNA, has been examined by alkaline sucrose gradient centrifugation of lysates of induced cells in order to study λ prophage excision. Thermal derepression of the prophage results in loss of F′450(λ) covalent circles, which is mediated by systems involved in excision and initiation of replication. When protocols known to result in prophage curing are used, the F′450(λ) is converted to an F′450 and a λ covalent circle; in normal excision leading to phage development, F′450 covalent circles are not found. We have shown that: (1) excision usually occurs later than initiation of DNA replication of the prophage so that the excised prophage is usually already replicated or in the act of replication; (2) the DNA growing points of the prophage leave the prophage and enter the bacterial DNA; (3) the int and xis genes are involved in the earliest detectable stage of the excision process, i.e. breakage of the DNA at the attachment region; (4) the xis gene product is involved in a weak non-specific nuclease activity in addition to its highly specific activity in excision; and (5) the excision system fails to attack a single attachment site.     

Genes from the exo–xis region of λ and Shiga toxin-converting bacteriophages influence lysogenization and prophage induction

The exo–xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ?24_Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ?24_Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo–xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo–xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.     

The Multidrug Resistance IncA/C Transferable Plasmid Encodes a Novel Domain-swapped Dimeric Protein-disulfide Isomerase

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (−161 mV) is more reducing than EcDsbC (−130 mV) and EcDsbG (−126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.